engleski

Comparison of 2-aminobenzamide, procainamide and RapiFluor-MS as derivatizing agents for high-throughput N-glycan analysis

Rising awareness of the universal importance of protein N-glycosylation governs the development of further advances in N-glycan analysis. Techniques wherein derivatized N-glycans are profiled using hydrophilic interaction chromatography (HILIC) with fluorescence (FLR) and mass spectrometry (MS) detection are now routinely performed in a high-throughput manner. Therefore, we aimed to examine the performance of frequently used labeling compounds – 2-aminiobenzamide (2-AB) and procainamide (ProA), and the recently introduced RapiFluor-MS (RF-MS) fluorescent tag. In all experiments N-glycans were released by PNGase F, fluorescently derivatized, purified by HILIC solid phase extraction and profiled using HILIC-UPLC-FLR-MS. We assessed sensitivity, linear range, limit of quantification, repeatability and labeling efficiency for all three labels. We also tested the performance of all three labels in a high- throughput setting on 68 different IgG samples, all in duplicates, and 22 identical IgG standards. In general, ProA labeled glycans had the highest FLR sensitivity (15-fold and 4-fold higher signal intensities compared to 2-AB and RF-MS, respectively) and RF-MS had the highest MS sensitivity (68-fold and 2-fold higher signal intensities compared to 2-AB and ProA, respectively). ProA and RF-MS showed comparable limits of quantification with both FLR and MS detection, whilst 2-AB exhibited the lowest sensitivity. All labeling procedures showed good and comparable repeatability. In conclusion, all three labels are a good choice for N-glycan derivatization in high-throughput HILIC-UPLC-FLR-MS N-glycan analysis, although ProA and RF-MS are a better option when higher sensitivity is needed.

2-aminobenzamide ; procainamide ; RapiFluor-MS ; glycosylation ; derivatization ; fluorescence ; HILIC ; MS

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